Delley2021

This method does not have a name. It was published in Scientific Reports: Modular barcode beads for microfluidic single cell genomics [Delley2021].. This method builds the foundation for PIP-seq. The complete full list of oligo sequences can be found in the Supplementary Tables from the Delley2021 paper: 41598_2021_90255_MOESM4_ESM.xlsx.

As the paper suggested, this is not really about scRNA-seq or any specific assays. Instead, it provides a modular procedures to prepare barcoded beads for high-throughput droplet single-cell assays for anything you are interested in (RNA, DNA, chromatin accessibility etc.). The point here is that you use Steps (1) - (7) described below to prepare bead barcodes. In the last step, you use pBB4 and pBB5 to "functionalise" the beads depending on what you need. If you want to investigate gene expressions, your pBB5 should contains UMI and oligo-dT, which is demonstrate on this page. If you are interested in chromatin accessibility, you can change pBB5 by putting Nextera sequence on it. You can customise pBB5 based on your own need.


Adapter and primer sequences:

pBB1: 5'- /5Acryd/ACTAACAATAAGCTCUAUCGATGACCTAATACGACTCACTATAGGGACAAATGCCGATTCCTGCTGAAC -3'

pBB2: 5'- /5Phos/TGACGTTCAGCAGGAATCGGCATTTGTCCCTATAGTGAGTCGTATTAGGTCATCGATAGAG -3'

plate-1-BC (this is Supp. Table 1): 5'- /5Phos/GTCA[0-3 bp Spacer][8-bp barcode1]AACC -3'

plate-1-SP (this is Supp. Table 2): 5'- /5Phos/[8-bp barcode1 rc][0-3 bp Spacer rc] -3'

plate-2-BC (this is Supp. Table 3): 5'- /5Phos/CTGT[8-bp barcode2 rc]GGTT -3'

plate-2-SP (this is Supp. Table 4): 5'- /5Phos/[8-bp barcode2] -3'

plate-3-BC (this is Supp. Table 5): 5'- /5Phos/ACAG[8-bp barcode3]CCTA -3'

plate-3-Sp (this is Supp. Table 6): 5'- /5Phos/[8-bp barcode3 rc] -3'

pBB4: 5'- CTCGAATAGG -3'

pBB5: 5'- /5Phos/TTCGAG[8-bp UMI]TTTTTTTTTTTTTTTTTTTTTTTTV -3'

* Plain barcodes (96) can be found here: delley2021_barcode.txt. The actual cell barcodes are the combinations of the same set of 96 barcodes.

Step-by-step generation of barcoded beads:

(1) Anneal plate-1-BC with plate-1-SP:


5'- GTCA[0-3 bp Spacer][8-bp barcode1]AACC - 3'
    3'- [0-3 bp Spacer][8-bp barcode1] -5'

(2) Anneal plate-2-BC with plate-2-SP:


    5'- [8-bp barcode2] -3'
3'- TTGG[8-bp barcode2]TGTC -5'

(3) Anneal plate-3-BC with plate-3-SP:


5'- ACAG[8-bp barcode3]CCTA -3'
    3'- [8-bp barcode3] -5'

(4) Form dissolvable acrylamide gel beads together with pBB1 and anneal pBB2 to the bead oligo:


|--5'- /5Acryd/ACTAACAATAAGCTCUAUCGATGACCTAATACGACTCACTATAGGGACAAATGCCGATTCCTGCTGAAC     -3'
                       3'- GAGATAGCTACTGGATTATGCTGAGTGATATCCCTGTTTACGGCTAAGGACGACTTGCAGT -5'

(5) Split the gel beads into wells and add annealed plate-1 for barcode1 ligation:


|--5'- /5Acryd/ACTAACAATAAGCTCUAUCGATGACCTAATACGACTCACTATAGGGACAAATGCCGATTCCTGCTGAACGTCA[0-3 bp Spacer][8-bp barcode1]AACC
                       3'- GAGATAGCTACTGGATTATGCTGAGTGATATCCCTGTTTACGGCTAAGGACGACTTGCAGT[0-3 bp Spacer][8-bp barcode1] -5'

(6) Pooling and redistribute to a new 96-well plate and add annealed plate-2 for barcode2 ligation:


|--5'- /5Acryd/ACTAACAATAAGCTCUAUCGATGACCTAATACGACTCACTATAGGGACAAATGCCGATTCCTGCTGAACGTCA[0-3 bp Spacer][8-bp barcode1]AACC[8-bp barcode2]
                       3'- GAGATAGCTACTGGATTATGCTGAGTGATATCCCTGTTTACGGCTAAGGACGACTTGCAGT[0-3 bp Spacer][8-bp barcode1]TTGG[8-bp barcode2]TGTC -5'

(7) Pooling and redistribute to a new 96-well plate and add annealed plate-3 for barcode3 ligation:


|--5'- /5Acryd/ACTAACAATAAGCTCUAUCGATGACCTAATACGACTCACTATAGGGACAAATGCCGATTCCTGCTGAACGTCA[0-3 bp Spacer][8-bp barcode1]AACC[8-bp barcode2]ACAG[8-bp barcode3]CCTA
                       3'- GAGATAGCTACTGGATTATGCTGAGTGATATCCCTGTTTACGGCTAAGGACGACTTGCAGT[0-3 bp Spacer][8-bp barcode1]TTGG[8-bp barcode2]TGTC[8-bp barcode3] -5'

(8) Add pBB4 and pBB5 to add RT primer to the beads:


|--5'- /5Acryd/ACTAACAATAAGCTCUAUCGATGACCTAATACGACTCACTATAGGGACAAATGCCGATTCCTGCTGAACGTCA[0-3 bp Spacer][8-bp barcode1]AACC[8-bp barcode2]ACAG[8-bp barcode3]CCTATTCGAG[8-bp UMI]TTTTTTTTTTTTTTTTTTTV
                       3'- GAGATAGCTACTGGATTATGCTGAGTGATATCCCTGTTTACGGCTAAGGACGACTTGCAGT[0-3 bp Spacer][8-bp barcode1]TTGG[8-bp barcode2]TGTC[8-bp barcode3]GGATAAGCTC -5'

(9) Denature by NaOH and get rid of the bottom strand. The beads oligos are ready to use for experiments:


|--5'- /5Acryd/ACTAACAATAAGCTCUAUCGATGACCTAATACGACTCACTATAGGGACAAATGCCGATTCCTGCTGAACGTCA[0-3 bp Spacer][8-bp barcode1]AACC[8-bp barcode2]ACAG[8-bp barcode3]CCTATTCGAG[8-bp UMI]TTTTTTTTTTTTTTTTTTTV -3'